Human IL-1 epsilon DNA and polypeptides

ABSTRACT

The invention is directed to purified and isolated novel human IL-1 epsilon polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, the use of such polypeptides and fragmented peptides in cellular and immune reactions and as molecular weight markers, the use of such polypeptides and fragmented peptides as controls for peptide fragmentation, and kits comprising these reagents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of 09/763,498, filed May 15, 2001,which is the National Stage of International Application No.PCT/US99/18771, filed Aug. 20, 1999, which hereby claims the benefit ofU.S. provisional applications Nos. 60/097,413, 60/098,595, and.60/099,974, filed Aug. 21, 1998, Aug. 31, 1998, and Sep. 11, 1998,respectively. The entire disclosures of these applications are reliedupon and incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to purified and isolated novel human IL-1epsilon polypeptides, the nucleic acids encoding such polypeptides,processes for production of recombinant forms of such polypeptides,antibodies generated against these polypeptides, fragmented peptidesderived from these polypeptides, the use of such polypeptides andfragmented peptides in cellular and immune reactions and as molecularweight markers, the use of such polypeptides and fragmented peptides ascontrols for peptide fragmentation, and kits comprising these reagents.

2. Description of Related Art

Interleukin-1 (IL-1) is a member of a large group of cytokines whoseprimary function is to mediate immune and inflammatory responses. Thereare five known IL-1 family members which include IL-1 alpha (IL-1α),IL-1 beta (IL-1α), IL-1 receptor antagonist (IL-1ra), IL-1 delta (IL-1δ,as disclosed in PCT US/99/00514), and IL-18 (previously known as IGIFand sometimes IL-1 gamma). IL-1 that is secreted by macrophages isactually a mixture of mostly IL-1β and some IL-1α (Abbas et al., 1994).IL-1α and IL-1β, which are first produced as 33 kD precursors that lacka signal sequence, are further processed by proteolytic cleavage toproduce secreted active forms, each about 17 kD. Additionally, the 33 kDprecursor of IL-1α is also active. Both forms of IL-1 are the productsof two different genes located on chromosome 2. Although the two formsare less than 30 percent homologous to each other, they both bind to thesame receptors and have similar activities.

IL-1ra, a biologically inactive form of IL-1, is structurally homologousto IL-1 and binds to the same receptors. Additionally, IL-1ra isproduced with a signal sequence which allows for efficient secretioninto the extracellular region where it competitively competes with IL-1(Abbas et al., 1994).

The IL-1 family ligands bind to two IL-1 receptors that are members ofthe Ig superfamily. IL-1 receptors include the 80 kDa type I receptor(IL-1RI) and a 68 kDa type II receptor (IL-1RII). The ligands also bindto a soluble proteolytic fragment of IL-1RII (sIL-1RII) (Colotta et al.,Science 261(5120):472-75, 1993).

The major source of IL-1 is the activated macrophage or mononuclearphagocyte. Other cells that produce IL-1 include epithelial andendothelial cells (Abbas et al., 1994). IL-1 secretion from macrophagesoccurs after the macrophage encounters and ingests gram-negativebacteria. Such bacteria contain lipopolysaccharide (LPS) molecules, alsoknown as endotoxin, in the bacterial cell wall. LPS molecules are theactive components that stimulate macrophages to produce tumor necrosisfactor (TNF) and IL-1. In this case, IL-1 is produced in response to LPSand TNF production. At low concentrations, LPS stimulates macrophagesand activates B-cells and other host responses needed to eliminate thebacterial infection; however, at high concentrations, LPS can causesevere tissue damage, shock, and even death.

The biological functions of IL-1 include activating vascular endothelialcells and lymphocytes, local tissue destruction, and fever (Janeway etal., 1996). At low levels, IL-1 stimulates macrophages and vascularendothelial cells to produce IL-6, upregulates molecules on the surfaceof vascular endothelial cells to increase leukocyte adhesion, andindirectly activates inflammatory leukocytes by stimulating mononuclearphagocytes and other cells to produce certain chemokines that activateinflammatory leukocytes. These IL-1 functions are crucial during lowlevel microbial infections. However, if the microbial infectionescalates, IL-1 acts systemically by inducing fever, stimulatingmononuclear phagocytes to produce IL-1 and IL-6, increasing theproduction of serum proteins from hepatocytes, and activating thecoagulation system. It is also known that IL-1 does not causehemorrhagic necrosis of tumors or suppress bone marrow stem celldivision. Nevertheless, IL-1 is lethal to humans at high concentrations.

Given the important function of IL-1, there is a need in the art foradditional members of the IL-1 ligand family. In addition, in view ofthe continuing interest in protein research and the immune system, thediscovery, identification, and roles of new proteins, such as human IL-1epsilon and its receptors, are at the forefront of modern molecularbiology and biochemistry. Despite the growing body of knowledge, thereis still a need in the art for the identity and function of proteinsinvolved in cellular and immune responses.

In yet another aspect of the invention, the identification of theprimary structure, or sequence, of an unknown sample protein is theculmination of an arduous process of experimentation. In order toidentify an unknown protein sample, the investigator can rely upon acomparison of the unknown protein sample to known peptides using avariety of techniques known to those skilled in the art. For instance,proteins are routinely analyzed using techniques such aselectrophoresis, sedimentation, chromatography, and mass spectrometry.

Comparison of an unknown protein sample to polypeptides of knownmolecular weight allows a determination of the apparent molecular weightof the unknown protein sample (T. D. Brock and M. T. Madigan, Biology ofMicroorganisms 76-77 (Prentice Hall, 6^(th) ed. 1991)). Proteinmolecular weight standards are commercially available to assist in theestimation of molecular weights of unknown protein samples (New EnglandBiolabs Inc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No.5,449,758). However, the molecular weight standards may not correspondclosely enough in size to the unknown sample protein to allow anaccurate estimation of apparent molecular weight. The difficulty inestimation of molecular weight is compounded in the case of proteinsthat are subjected to fragmentation by chemical or enzymatic means (A.L. Lehninger, Biochemistry 106-108 (Worth Books, 2d ed. 1981)).

The unique nature of the composition of a protein with regard to itsspecific amino acid constituents results in a unique positioning ofcleavage sites within the protein. Specific fragmentation of a proteinby chemical or enzymatic cleavage results in a unique “peptidefingerprint” (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106,1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980). Consequently,cleavage at specific sites results in reproducible fragmentation of agiven protein into peptides of precise molecular weights. Furthermore,these peptides possess unique charge characteristics that determine theisoelectric pH of the peptide. These unique characteristics can beexploited using a variety of electrophoretic and other techniques (T. D.Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall,6d ed. 1991)).

When a peptide fingerprint of an unknown protein is obtained, this canbe compared to a database of known proteins to assist in theidentification of the unknown protein (W. J. Henzel et al., Proc. Natl.Acad. Sci. USA 90:5011-5015, 1993; B. Thiede et al., Electrophoresis1996, 17:588-599, 1996). A variety of computer software programs areaccessible via the Internet to the skilled artisan for the facilitationof such comparisons, such as MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site:www.mann.embl-heiedelberg.de . . . deSearch/FR_PeptideSearchForm.html),and ProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programsallow the user to specify the cleavage agent and the molecular weightsof the fragmented peptides within a designated tolerance. The programscompare these molecular weights to protein databases to assist in theelucidation of the identity of the sample protein. Accurate informationconcerning the number of fragmented peptides and the precise molecularweight of those peptides is required for accurate identification.Therefore, increasing the accuracy in the determination of the number offragmented peptides and the precise molecular weight of those peptidesshould result in enhanced success in the identification of unknownproteins.

Fragmentation of proteins is further employed for the production offragments for amino acid composition analysis and protein sequencing (P.Matsudiara, J. Biol. Chem. 262:10035-10038, 1987; C. Eckerskorn et al.,Electrophoresis 1988, 9:830-838, 1988), particularly the production offragments from proteins with a “blocked” N-terminus. In addition,fragmentation of proteins can be used in the preparation of peptides formass spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA90:5011-5015, 1993; B. Thiede et al., Electrophoresis 1996, 17:588-599,1996), for immunization, for affinity selection (R. A. Brown, U.S. Pat.No. 5,151,412), for determination of modification sites (e.g.phosphorylation), for generation of active biological compounds (T. D.Brock and M. T. Madigan, Biology of Microorganisms 300-301 (PrenticeHall, 6^(th) ed. 1991)), and for differentiation of homologous proteins(M. Brown et al., J. Gen. Virol. 50:309-316, 1980).

Thus, there also exists a need in the art for IL-1 polypeptides suitablefor use in peptide fragmentation studies, for use in molecular weightmeasurements, and for use in protein sequencing using tandem massspectrometry.

SUMMARY OF THE INVENTION

The invention aids in fulfilling these needs in the art by providingisolated human IL-1 epsilon nucleic acids and polypeptides encoded bythese nucleic acids. Specifically, the invention encompasses an isolatedhuman IL-1 epsilon nucleic acid molecule comprising the DNA sequences ofSEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12 and an isolated human IL-1epsilon nucleic acid molecule encoding the amino acid sequence of SEQ IDNO:6, SEQ ID NO:8, and SEQ ID NO:13, as well as nucleic acid moleculescomplementary to these sequences. Both single-stranded anddouble-stranded RNA and DNA nucleic acid molecules are encompassed bythe invention, as well as nucleic acid molecules that hybridize to adenatured, double-stranded DNA relating to SEQ ID NO:5, SEQ ID NO:7, orSEQ ID NO:12. Also encompassed are isolated nucleic acid molecules thatare derived by in vitro mutagenesis from SEQ ID NO:5, SEQ ID NO:7, orSEQ ID NO:12, are degenerate from SEQ ID NO:5, SEQ ID NO:7, or SEQ IDNO:12, are allelic variants of human DNA of the invention, and arespecies homologs of DNA of the invention. The invention also encompassesrecombinant vectors that direct the expression of these nucleic acidmolecules and host cells transformed or transfected with these vectors.In addition, the invention encompasses methods of using the nucleic acidnoted above in assays to identify chromosomes, map human genes, andstudy the immune system.

The invention also encompasses isolated polypeptides encoded by thesenucleic acid molecules, synthetic polypeptides encoded by these nucleicacid molecules, and peptides and fragments of these polypeptides.Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention. The inventionfurther encompasses methods for the production of IL-1 epsilonpolypeptides, including culturing a host cell under conditions promotingexpression and recovering the polypeptide from the culture medium.Especially, the expression of IL-1 epsilon polypeptides in bacteria,yeast, plant, insect, and animal cells is encompassed by the invention.

In general, the polypeptides of the invention can be used to studycellular processes such as immune regulation, cell proliferation, celldeath, and inflammatory responses. In addition, the IL-1 epsilon ligandpolypeptides, related IL-1 epsilon polypeptides, and fragments thereof,can be used to identify proteins associated with IL-1-like ligands andIL-1-like receptors.

In addition, assays utilizing IL-1 epsilon ligand polypeptides, relatedIL-1 epsilon polypeptides, and fragments thereof to screen for potentialinhibitors of activity associated with polypeptide counter-structuremolecules, and methods of using IL-1 epsilon ligand polypeptides,related IL-1 epsilon polypeptides, and fragments thereof as therapeuticagents for the treatment of diseases mediated by IL-1 epsilon ligandpolypeptide counter-structure molecules are encompassed by theinvention. Further, methods of using IL-1 epsilon ligand polypeptides,related IL-1 epsilon polypeptides, and fragments thereof in the designof inhibitors thereof are also an aspect of the invention.

The invention further includes a method for using these polypeptides andfragmented peptides thereof as molecular weight markers that allow theestimation of the molecular weight of a protein or a fragmented proteinsample, as well as a method for the visualization of the molecularweight markers of the invention thereof using electrophoresis. Theinvention further encompasses methods for using the polypeptides of theinvention and fragmented peptides thereof as markers for determining theisoelectric point of a sample protein, as well as controls forestablishing the extent of fragmentation of a protein sample.

Further encompassed by this invention are kits to aid in thesedeterminations.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIG. 1 is the nucleotide sequences of human IL-1 epsilon DNA of theinvention, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12.

FIG. 2 is the amino acid sequence of polypeptides, SEQ ID NO:6, SEQ IDNO:8, and SEQ ID NO:13, encoded by the nucleotide sequences of SEQ IDNO:5, SEQ ID NO:7, and SEQ ID NO:12, respectively.

FIG. 3 depicts the amino acid homology between the 3′ exon of human IL-1epsilon and murine IL-1 epsilon (long form).

FIG. 4 depicts the amino acid homology between human IL-1 epsilon (aminoacids 51-159) and murine IL-1 epsilon (long form).

DETAILED DESCRIPTION OF THE INVENTION

Interleukin-1 (IL-1) receptors are members of the large Ig superfamilyof cytokine receptors, many of which mediate the response of immunesystem cells, in particular lymphocytes. In recent years, members of thefamily of ligands that bind to these receptors have been discovered atan accelerated pace. The increase in the number of known IL-1 ligandshas been largely due to the advent of gene cloning and sequencingtechniques. Amino acid sequences deduced from nucleotide sequences areconsidered to represent IL-1 ligands if they share homology with otherknown IL-1 ligands.

Mouse IL-1 epsilon is a homolog of the known IL-1 genes, IL-1α, IL-1β,IL-1δ (disclosed in PCT US/99/00514) and IL-1ra, and more recently,IL-18, previously known sometimes as IL-1 gamma. Mouse IL-1 epsilon wasfirst identified by searching the EST database, and discovering an ESTcorresponding to mouse IL-1 epsilon (accession number AA030324). Theentire open reading frame for the “long form” (see below) is containedin this EST.

Mouse IL-1 Epsilon (Long Form) DNA Sequence ATGTTCAGGA TCTTAGTAGTCGTGTGTGGA TCCTGCAGAA CAATATCCTC (SEQ ID NO:1) ACTGCAGTCC CAAGGAAAGAGCAAACAGTT CCAGGAAGGG AACATAATGG AAATGTACAA CAAAAAGGAA CCTGTAAAAGCCTCTCTCTT CTATCACAAG AAGAGTGGTA CAACCTCTAC ATTTGAGTCT GCAGCCTTCCCTGGTTGGTT CATCGCTGTC TGCTCTAAAG GGAGCTGCCC ACTCATTCTG ACCCAAGAACTGGGGGAAAT CTTCATCACT GACTTCGAGA TGATTGTGGT ACATTAA

Mouse IL-1 Epsilon (Long Form) Amino Acid Sequence MFRILVVVCG SCRTISSLQSQGKSKQFQEG NIMEMYNKKE PVKASLFYHK (SEQ ID NO:2) KSGTTSTFES AAFPGWFIAVCSKGSCPLIL TQELGEIFIT DFEMIVVH*

While showing homology to the IL-1 genes, mouse IL-1 epsilon is unusualin that the EST originally identified appeared to encode the C-terminaltwo-thirds of an IL-1-like molecule. In addition, during studies of theexpression of IL-1 epsilon, it became apparent that there are two,alternatively spliced, forms of mRNA that encode proteins with identicalN-termini but divergent C-termini. The longer of these two proteins wasthat encoded by the original EST. The shorter (sometimes called the“isoform”) is approximately one-third the length of a typical IL-1family molecule.

Mouse IL-1 Epsilon (Short Form) DNA Sequence ATGTTCAGGA TCTTAGTAGTCGTGTGTGGA TCCTGCAGAA CAATATCCTC (SEQ ID NO:3) ACTGCAGTCC CAAGGAAAGAGCAAACAGTT CCAGTCACTA TTACCTTGCT CCCATGCCAA TATCTGGACA CTCTTGAGACGAACAGGGGG GATCCCACGT ACATGGGAGT GCAAAGGCCG ATGA

Mouse IL-1 Epsilon (Short Form) Amino Acid Sequence MFRILVVVCGSCRTISSLQS QGKSKQFQSL LPCSHANIWT LLRRTGGIPR (SEQ ID NO:4) TWECKGR*

These two proteins (the long and the short form), encoded byalternatively spliced versions of the same original RNA transcript, mayassociate non-covalently and thus form a “whole” IL-1-like molecule.

In any event, using as a probe the mixed cDNAs for mouse long-form andshort-form IL-1 epsilon, human IL-1 epsilon has been identified byscreening of a human genomic library Sequencing of a clone obtained fromthe human genomic library reveals a stretch of DNA which contains anopen reading frame, encoding a portion of a protein with high homologyto mouse IL-1 epsilon in the same region. The open reading frame appearsto be an exon (the 3′ most exon of the coding region). The spliceacceptor site at the 5′ end of this exon is in the identical position tothe splice acceptor site of the corresponding exon in mouse IL-1epsilon.

The DNA and amino acid sequences of this exon corresponding to humanIL-1 epsilon are set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively.

Nucleotide Sequence of Human IL-1 Epsilon DNA: GAAAAGGATA TAATGGATTTGTACAACCAA CCCGAGCCTG TGAAGTCCTT (SEQ ID NO:5) TCTCTTCTAC CACAGCCAGAGTGGCAGGAA CTCCACCTTC GAGTCTGTGG CTTTCCCTGG CTGGTTCATC GCTGTCAGCTCTGAAGGAGG CTGTCCTCTC ATCCTTACCC AAGAACTGGG GAAAGCCAAC ACTACTGACTTTGGGTTAAC TATGCTGTTT TAA

A preferred polypeptide encoded by the nucleic acid sequence is setforth below:

Amino Acid Sequence of Human IL-1 Epsilon:

Translation in relevant reading frame (5′ 3′): EKDIMDLYNQ PEPVKSFLFYHSQSGRNSTF ESVAFPGWFI AVSSEGGCPL (SEQ ID NO:6) ILTQELGKAN TTDFGLTMLF *

The full-length human IL-1 epsilon DNA sequence was isolated asdescribed in Example I. The DNA and amino acid sequence of thefull-length human IL-1 epsilon are set forth in SEQ ID NO:7 and SEQ IDNO:8, respectively.

Full-Length Nucleotide Sequence of Human IL-1 Epsilon DNA: ATGGAAAAAGCATTGAAAAT TGACACACCT CAGCAGGGGA GCATTCAGGA (SEQ ID NO:7) TATCAATCATCGGGTGTGGG TTCTTCAGGA CCAGACGCTC ATAGCAGTCC CGAGGAAGGA CCGTATGTCTCCAGTCACTA TTGCCTTAAT CTCATGCCGA CATGTGGAGA CCCTTGAGAA AGACAGAGGGAACCCCATCT ACCTGGGCCT GAATGGACTC AATCTCTGCC TGATGTGTGC TAAAGTCGGGGACCAGCCCA CACTGCAGCT GAAGGAAAAG GATATAATGG ATTTGTACAA CCAACCCGAGCCTGTGAAGT CCTTTCTCTT CTACCACAGC CAGAGTGGCA GGAACTCCAC CTTCGAGTCTGTGGCTTTCC CTGGCTGGTT CATCGCTGTC AGCTCTGAAG GAGGCTGTCC TCTCATCCTTACCCAAGAAC TGGGGAAAGC CAACACTACT GACTTTGGGT TAACTATGCT GTTTTAAFull-Length Amino Acid Sequence of Human IL-1 Epsilon:

Translation in relevant reading frame (5′ to 3′): MEKALKIDTP QQGSIQDINHRVWVLQDQTL IAVPRKDRMS PVTIALISCR (SEQ ID NO:8) HVETLEKDRG NPIYLGLNGLNLCLMCAKVG DQPTLQLKEK DIMDLYNQPE PVKSFLFYHS QSGRNSTFES VAFPGWFIAVSSEGGCPLIL TQELGKANTT DFGLTMLF*

In addition, a single nucleotide polymorphism was identified in thehuman IL-1 epsilon gene. Specifically, the polymorphism comprises anadenosine to guanosine substitution at nucleotide 35. The polypeptideencoded by this polymorphic IL-1 epsilon gene has an arginine residue atposition 12 rather than a glutamine residue. The DNA sequence of thehuman IL-1 epsilon gene containing this single nucleotide polymorphismis set forth in SEQ ID NO:12, and the full-length amino acid sequencecorresponding to this polymorphic gene is set forth in SEQ ID NO:13.

Full-Length Nucleotide Sequence of Polymorphic Human IL-1 Epsilon DNA:ATGGAAAAAG CATTGAAAAT TGACACACCT CAGCGGGGGA GCATTCAGGA (SEQ ID NO:12)TATCAATCAT CGGGTGTGGG TTCTTCAGGA CCAGACGCTC ATAGCAGTCC CGAGGAAGGACCGTATGTCT CCAGTCACTA TTGCCTTAAT CTCATGCCGA CATGTGGAGA CCCTTGAGAAAGACAGAGGG AACCCCATCT ACCTGGGCCT GAATGGACTC AATCTCTGCC TGATGTGTGCTAAAGTCGGG GACCAGGCCA CACTGCAGCT GAAGGAAAAG GATATAATGG ATTTGTACAACCAACCCGAG CCTGTGAAGT CCTTTCTCTT CTACCACAGC CAGAGTGGCA GGAACTCCACCTTCGAGTCT GTGGCTTTCC CTGGCTGGTT CATCGCTGTC AGCTCTGAAG GAGGCTGTCCTCTCATCCTT ACCCAAGAAC TGGGGAAAGC CAACACTACT GACTTTGGGT TAACTATGCTGTTTTAAFull-Length Amino Acid Sequence of Human IL-1 Epsilon:

Translation in relevant reading frame (5′ to 3′): MEKALKIDTP QRGSIQDINHRVWVLQDQTL IAVPRKDRMS PVTIALISCR (SEQ ID NO:13) HVETLEKDRG NPIYLGLNGLNLCLMCAKVG DQPTLQLKEK DIMDLYNQPE PVKSFLFYHS QSGRNSTFES VAFPGWFIAVSSEGGCPLIL TQELGKANTT DFGLTMLF*

The discovery of this DNA encoding IL-1 epsilon enables the constructionof expression vectors comprising nucleic acid sequences encoding IL-1epsilon polypeptides of the invention; host cells transfected ortransformed with the expression vectors; biologically active human IL-1epsilon polypeptides and molecular weight markers as isolated andpurified proteins; and antibodies immunoreactive with polypeptides ofthe invention.

Nucleic Acid Molecules

In a particular embodiment, the invention relates to certain isolatednucleotide sequences. A “nucleotide sequence” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct, that has been derived from DNA or RNAisolated at least once in substantially pure form (i.e., free ofcontaminating endogenous materials) and in a quantity or concentrationenabling identification, manipulation, and recovery of its componentnucleotide sequences by standard biochemical methods (such as thoseoutlined in Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989)). Such sequences are preferably provided and/or constructed inthe form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, that are typically present ineukaryotic genes. Sequences of non-translated DNA can be present 5′ or3′ from an open reading frame, where the same do not interfere withmanipulation or expression of the coding region.

Particularly preferred nucleotide sequences of the invention are SEQ IDNO:5, SEQ ID NO:7, and SEQ ID NO:12, as set forth above. The inventionfurther encompasses isolated fragments and oligonucleotides derived fromthe nucleotide sequences of SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12.Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native nucleotidesequences disclosed herein under conditions of moderate or severestringency, and which encode polypeptides or fragments thereof of theinvention. These isolated DNA and RNA sequences also include full lengthDNA or RNA molecules encoding for IL-1 epsilon polypeptides.

As used herein, conditions of moderate stringency, as known to thosehaving ordinary skill in the art, and as defined by Sambrook et al.Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Vol. 1, pp.1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use of aprewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6×SSC at42EC (or other similar hybridization solution, such as Stark's solution,in 50% formamide at 42EC), and washing conditions of about 60EC,0.5×SSC, 0.1% SDS. Conditions of high stringency are defined ashybridization conditions as above, and with washing at 68EC, 0.2×SSC,0.1% SDS. The skilled artisan will recognize that the temperature andwash solution salt concentration can be adjusted as necessary accordingto factors such as the length of the probe.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence can vary from thatshown in SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:12, and still encode apolypeptide having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8,or SEQ ID NO:13, respectively. Such variant DNA sequences can resultfrom silent mutations (e.g., occurring during PCR amplification) or canbe the product of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA derived from thecoding region of a native mammalian gene; (b) cDNA comprising thenucleotide sequence of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:12; (c)DNA encoding the polypeptides of SEQ ID NO:6, SEQ ID NO:8, or SEQ IDNO:13; (d) DNA capable of hybridization to a DNA of (a), (b), or (c)under conditions of moderate stringency and which encodes polypeptidesof the invention; and (e) DNA which is degenerate as a result of thegenetic code to a DNA defined in (a), (b), (c), or (d) and which encodespolypeptides of the invention. Of course, polypeptides encoded by suchequivalent DNA sequences are encompassed by the invention.

DNA that is equivalent to the DNA sequence of SEQ ID NO:5, SEQ ID NO:7,or SEQ ID NO:12 will hybridize under moderately stringent conditions tothe double-stranded native DNA sequence that encode polypeptidescomprising amino acid sequences of SEQ ID NO:6, SEQ ID NO:8, or SEQ IDNO:13. Examples of polypeptides encoded by such DNA, include, but arenot limited to, polypeptide fragments and polypeptides comprisinginactivated N-glycosylation site(s), inactivated protease processingsite(s), or conservative amino acid substitution(s), as described below.Polypeptides encoded by DNA derived from other mammalian species,wherein the DNA will hybridize to the complement of the DNA of SEQ IDNO:5, SEQ ID NO:7, or SEQ ID NO:12, are also encompassed.

Expression

The nucleic acid sequence encoding polypeptides of the invention can beinserted into recombinant expression vectors using well known methods.The expression vectors include a DNA sequence of the invention operablylinked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequence functionally relates tothe DNA sequence of the invention. Thus, a promoter nucleotide sequenceis operably linked to a DNA sequence if the promoter nucleotide sequencecontrols the transcription of the DNA sequence of the invention. Theability to replicate in the desired host cells, usually conferred by anorigin of replication, and a selection gene by which transformants areidentified can additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with polypeptides of the invention can beincorporated into expression vectors. For example, a DNA sequence for asignal peptide (secretory leader) can be fused in-frame to thenucleotide sequence of the invention so that the polypeptide isinitially translated as a fusion protein comprising the signal peptide.A signal peptide that is functional in the intended host cells enhancesextracellular secretion of the polypeptide. The signal peptide can becleaved from the polypeptide upon secretion of polypeptide from thecell.

Suitable host cells for expression of polypeptides of the inventioninclude prokaryotes, yeast or higher eukaryotic cells. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described, for example, in Pouwels etal. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985).Cell-free translation systems could also be employed to producepolypeptides of the invention using RNAs derived from DNA constructsdisclosed herein.

Prokaryotic Systems

Prokaryotes include gram negative or gram positive organisms. Suitableprokaryotic host cells for transformation include, for example,Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and variousother species within the genera Bacillus, Pseudomonas, Streptomyces, andStaphylococcus. In a prokaryotic host cell, such as Escherichia coli, apolypeptide of the invention can include an N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in theprokaryotic host cell. The N-terminal Met can be cleaved from theexpressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells also generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. To construct an expression vectorusing pBR322, an appropriate promoter and a DNA sequence of theinvention are inserted into the pBR322 vector.

Other commercially available vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec,Madison, Wis., USA). Other commercially available vectors include thosethat are specifically designed for the expression of proteins; thesewould include pMAL-p2 and pMAL-c2 vectors that are used for theexpression of proteins fused to maltose binding protein (New EnglandBiolabs, Beverly, Mass., USA).

The promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include ∃-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage 8 P_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection, which incorporate derivatives of the 8P_(L) promoter, include plasmid pHUB2 (resident in E. coli strain JMB9(ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

The DNA of the invention can be cloned in-frame into the multiplecloning site of an ordinary bacterial expression vector. Ideally thevector contains an inducible promoter upstream of the cloning site, suchthat addition of an inducer leads to high-level production of therecombinant protein at a time of the investigator's choosing. For someproteins, expression levels can be boosted by incorporation of codonsencoding a fusion partner (such as hexahistidine) between the promoterand the gene of interest.

For expression of the recombinant protein, the bacterial cells arepropagated in growth medium until reaching a pre-determined opticaldensity. Expression of the recombinant protein is then induced, e.g., byaddition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activatesexpression of proteins from plasmids containing a lac operator/promoter.After induction (typically for 1-4 hours), the cells are harvested bypelleting in a centrifuge, e.g. at 5,000×G for 20 minutes at 4EC.

For recovery of the expressed protein, the pelleted cells may beresuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and thenpassed two or three times through a French press. Most highly-expressedrecombinant proteins form insoluble aggregates known as inclusionbodies. Inclusion bodies can be purified away from the soluble proteinsby pelleting in a centrifuge at 5,000×G for 20 minutes, 4EC. Theinclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% TritonX-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT.Any material that cannot be dissolved is removed by centrifugation(10,000×G for 20 minutes, 20EC). The protein of interest will, in mostcases, be the most abundant protein in the resulting clarifiedsupernatant. This protein may be “refolded” into the active conformationby dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl₂/5 mM Zn(OAc)₂/1 mMGSSG/0.1 mM GSH. After refolding, purification can be carried out by avariety of chromatographic methods such as ion exchange or gelfiltration. In some protocols, initial purification may be carried outbefore refolding. As an example, hexahistidine-tagged fusion proteinsmay be partially purified on immobilized nickel.

While the preceding purification and refolding procedure assumes thatthe protein is best recovered from inclusion bodies, those skilled inthe art of protein purification will appreciate that many recombinantproteins are best purified out of the soluble fraction of cell lysates.In these cases, refolding is often not required, and purification bystandard chromatographic methods can be carried out directly.

Yeast Systems

Polypeptides of the invention alternatively can be expressed in yeasthost cells, preferably from the Saccharomyces genus (e.g., S.cerevisiae). Other genera of yeast, such as Pichia, K. lactis, orKluyveromyces, can also be employed. Yeast vectors will often contain anorigin of replication sequence from a 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980), or otherglycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; andHolland et al., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van denBerg et. al., Bio/Technology, 8:135-139 (1990). Another alternative isthe glucose-repressible ADH2 promoter described by Russell et al. (J.Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982).Shuttle vectors replicable in both yeast and E. coli can be constructedby inserting DNA sequences from pBR322 for selection and replication inE. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast ∀-factor leader sequence can be employed to direct secretionof a polypeptide of the invention. The ∀-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274.Other leader sequences suitable for facilitating secretion ofrecombinant polypeptides from yeast hosts are known to those of skill inthe art. A leader sequence can be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine, and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence can be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian and Insect Systems

Alternatively, mammalian or insect host cell culture systems can beemployed to express recombinant polypeptides of the invention.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).Established cell lines of mammalian origin also can be employed.Examples of suitable mammalian host cell lines include the COS-7 line ofmonkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981),L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary(CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and theCV-1/EBNA-1 cell line (ATCC CRL 10478) derived from the African greenmonkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al.(EMBO J. 10: 2821, 1991). Established methods for introducing DNA intomammalian cells have been described (Kaufman, R. J., Large ScaleMammalian Cell Culture, 1990, pp. 15-69). Additional protocols usingcommercially available reagents, such as Lipofectamine (Gibco/BRL) orLipofectamine-Plus, can be used to transfect cells (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). In addition,electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in Sambrook et al. MolecularCloning: A Laboratory Manual, 2^(nd) ed. Vol. 1-3, Cold Spring HarborLaboratory Press, 1989. Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487-511, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B 11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529-534) and thetripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras etal., J. Biol. Chem. 257:13475-13491, 1982). The internal ribosome entrysite (IRES) sequences of viral origin allows dicistronic mRNAs to betranslated efficiently (Oh and Sarnow, Current Opinion in Genetics andDevelopment 3:295-300, 1993; Ramesh et al., Nucleic Acids Research24:2697-2700, 1996). Expression of a heterologous cDNA as part of adicistronic mRNA followed by the gene for a selectable marker (e.g.DHFR) has been shown to improve transfectability of the host andexpression of the heterologous cDNA (Kaufman, Meth. in Enzymology,1990). Exemplary expression vectors that employ dicistronic mRNAs arepTR—DC/GFP described by Mosser et al., Biotechniques 22:150-161, 1997,and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp.529-534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335-348, 1989. Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg(Mol. Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are describedin EP-A-0367566, and in U.S. patent application Ser. No. 07/701,415,filed May 16, 1991, incorporated by reference herein. The vectors can bederived from retroviruses. In place of the native signal sequence, aheterologous signal sequence can be added, such as the signal sequencefor IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence forIL-2 receptor described in Cosman et al., Nature 312:768 (1984); theIL-4 signal peptide described in EP 367,566; the type I IL-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIIL-1 receptor signal peptide described in EP 460,846.

Another useful expression vector, pFLAG, can be used. FLAG technology iscentered on the fusion of a low molecular weight (1 kD), hydrophilic,FLAG marker peptide to the N-terminus of a recombinant protein expressedby the pFLAG-1™ Expression Vector (1) (obtained from IBI Kodak).

Polypeptides of the Invention

As noted above, the present invention also includes isolated andpurified polypeptides. As used herein, the “polypeptides” of theinvention refers to a genus of polypeptides that further encompassesproteins having the amino acid sequence of SEQ ID NO: 6, SEQ ID NO:8, orSEQ ID NO:13, as well as those proteins having a high degree ofsimilarity (at least 90% homology) with such amino acid sequences andwhich proteins are biologically active. In addition, polypeptides of theinvention refers to the gene products of the nucleotides of SEQ ID NO:5,SEQ ID NO:7, and SEQ ID NO:12.

Isolation and Purification

The term “isolated and purified” as used herein, means that thepolypeptides or fragments of the invention are essentially free ofassociation with other proteins or polypeptides, for example, as apurification product of recombinant host cell culture or as a purifiedproduct from a non-recombinant source. The term “substantially purified”as used herein, refers to a mixture that contains polypeptides orfragments of the invention and is essentially free of association withother proteins or polypeptides, but for the presence of known proteinsthat can be removed using a specific antibody, and which substantiallypurified polypeptides or fragments thereof can be used as molecularweight markers. The term “purified” refers to either the “isolated andpurified” form of polypeptides of the invention or the “substantiallypurified” form of polypeptides of the invention, as both are describedherein.

An isolated and purified polypeptide according to the invention can beproduced by recombinant expression systems as described above orpurified from naturally occurring cells.

In one preferred embodiment, the expression of recombinant IL-1 epsilonpolypeptides can be accomplished utilizing fusions of sequences encodingIL-1 epsilon polypeptides to sequences encoding another polypeptide toaid in the purification of polypeptides of the invention. An example ofsuch a fusion is a fusion of sequences encoding an IL-1 epsilonpolypeptide to sequences encoding the product of the malE gene of thepMAL-c2 vector of New England Biolabs, Inc. Such a fusion allows foraffinity purification of the fusion protein, as well as separation ofthe maltose binding protein portion of the fusion protein from thepolypeptide of the invention after purification.

The insertion of DNA encoding the IL-1 epsilon polypeptide into thepMAL-c2 vector can be accomplished in a variety of ways using knownmolecular biology techniques. The preferred construction of theinsertion contains a termination codon adjoining the carboxyl terminalcodon of the polypeptide of the invention. In addition, the preferredconstruction of the insertion results in the fusion of the aminoterminus of the polypeptide of the invention directly to the carboxylterminus of the Factor Xa cleavage site in the pMAL-c2 vector. A DNAfragment can be generated by PCR using DNA of the invention as thetemplate DNA and two oligonucleotide primers. Use of the oligonucleotideprimers generates a blunt-ended fragment of DNA that can be isolated byconventional means. This PCR product can be ligated together withpMAL-p2 (digested with the restriction endonuclease Xmn I) usingconventional means. Positive clones can be identified by conventionalmeans. Induction of expression and purification of the fusion proteincan be performed as per the manufacturer's instructions and as notedabove. This construction facilitates a precise separation of thepolypeptide of the invention from the fused maltose binding proteinutilizing a simple protease treatment as per the manufacturer'sinstructions. In this manner, purified IL-1 epsilon polypeptide can beobtained. Furthermore, such a constructed vector can be easily modifiedusing known molecular biology techniques to generate additional fusionproteins. It is understood, of course, that many different vectors andtechniques, as noted above, can be used for the expression andpurification of polypeptides of the invention and that this embodimentin no way limits the scope of the invention.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells by any convenient method (includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents), centrifugation, extraction from cell pellets if aninsoluble polypeptide, or from the supernatant fluid if a solublepolypeptide, followed by one or more concentration, salting-out, ionexchange, affinity purification or size exclusion chromatography steps.As is known to the skilled artisan, procedures for purifying arecombinant protein will vary according to such factors as the type ofhost cells employed and whether or not the recombinant protein issecreted into the culture medium. For example, when expression systemsthat secrete the recombinant protein are employed, the culture mediumfirst can be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. Following the concentration step, the concentratecan be applied to a purification matrix such as a gel filtration medium.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel having pendantmethyl or other aliphatic groups) can be employed to further purify thepolypeptides. Some or all of the foregoing purification steps, invarious combinations, are well known and can be employed to provide anisolated and purified recombinant protein.

It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized.

In this aspect of the invention, polypeptide-binding proteins, such asthe anti-polypeptide antibodies of the invention or other proteins thatmay interact with the polypeptide of the invention, can be bound to asolid phase support such as a column chromatography matrix or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence ofpolypeptide-binding proteins of the invention to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

In the methods described above, suitable polypeptide-binding proteinsare anti-polypeptide antibodies, and other proteins that are capable ofhigh-affinity binding of polypeptides of the invention. A preferredpolypeptide-binding protein is an anti-polypeptide monoclonal antibody.

In a preferred embodiment, transformed yeast host cells are employed toexpress polypeptides of the invention as a secreted polypeptide in orderto simplify purification. Secreted recombinant polypeptide from a yeasthost cell fermentation can be purified by methods analogous to thosedisclosed by Urdal et al. (J. Chromatog. 296:171, 1984) (relating to theuse of two sequential, reversed-phase HPLC steps for purification).

Variants

The invention also includes variants of the polypeptides of theinvention. A polypeptide “variant” as referred to herein means apolypeptide substantially homologous to native polypeptides of theinvention, but which has an amino acid sequence different from that ofnative polypeptides (human, murine or other mammalian species) of theinvention because of one or more deletions, insertions or substitutions.The variant amino acid sequence preferably is at least 80% identical toa native polypeptide amino acid sequence. Also contemplated areembodiments in which a polypeptide or fragment comprises an amino acidsequence that is at least 90% identical, at least 95% identical, atleast 98% identical, at least 99% identical, or at least 99.9% identicalto the preferred polypeptide or fragment thereof. The percent identitycan be determined, for example, by comparing sequence information usingthe GAP computer program, version 6.0 described by Devereux et al.(Nucl. Acids Res. 12:387, 1984) and available from the University ofWisconsin Genetics Computer Group (UWGCG). The GAP program utilizes thealignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970),as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

Variants can comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Naturally occurring variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events, proteolytic cleavage of the IL-1 epsilonpolypeptides, or transcription/translation from different alleles.Variations attributable to proteolysis include, for example, differencesin the N or C-termini upon expression in different types of host cells,due to proteolytic removal of one or more terminal amino acids from thepolypeptides (generally from 1-5 terminal amino acids) of the invention.

Oligomers

The polypeptides of the invention can also exist as oligomers, such ascovalently linked or non-covalently linked dimers or trimers. Oligomerscan be linked by disulfide bonds formed between cysteine residues ondifferent polypeptides.

In one embodiment of the invention, a polypeptide dimer is created byfusing polypeptides of the invention to the Fc region of an antibody(e.g., IgG1) in a manner that does not interfere with the biologicalactivity of these polypeptides. The Fc region preferably is fused to theC-terminus of a soluble polypeptide of the invention, to form an Fcfusion or an Fc polypeptide. The terms “Fc fusion protein” or “Fcpolypeptides” as used herein includes native and mutein forms, as wellas truncated Fc polypeptides containing the hinge region that promotesdimerization. Exemplary methods of making Fc polypeptides set forthabove are disclosed in U.S. Pat. Nos. 5,426,048 and 5,783,672, both ofwhich are incorporated herein by reference.

General preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the polypeptide:Fcfusion protein of the invention is inserted into an appropriateexpression vector. Polypeptide:Fc fusion proteins are allowed toassemble much like antibody molecules, whereupon interchain disulfidebonds form between Fc polypeptides, yielding divalent polypeptides ofthe invention. If fusion proteins are made with both heavy and lightchains of an antibody, it is possible to form a polypeptide oligomerwith as many as four polypeptides extracellular regions. Alternatively,one can link two soluble polypeptide domains with a peptide linker.

Alterations

As stated above, the invention provides isolated and purifiedpolypeptides, and fragments thereof, both recombinant andnon-recombinant. Variants and derivatives of native polypeptides can beobtained by mutations of nucleotide sequences coding for nativepolypeptides. Alterations of the native amino acid sequence can beaccomplished by any of a number of conventional methods. Mutations canbe introduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes an analog having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462,all of which are incorporated by reference.

Polypeptides of the invention can also be modified to create polypeptidederivatives by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, polyethylene glycol (PEG)groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of polypeptides of the invention can be prepared by linkingthe chemical moieties to functional groups on polypeptide amino acidside chains or at the N-terminus or C-terminus of a polypeptide of theinvention or the extracellular domain thereof. Other derivatives ofpolypeptides within the scope of this invention include covalent oraggregative conjugates of these polypeptides or peptide fragments withother proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. For example, the conjugatecan comprise a signal or leader polypeptide sequence (e.g. the ∀-factorleader of Saccharomyces) at the N-terminus of a polypeptide of theinvention. The signal or leader peptide co-translationally orpost-translationally directs transfer of the conjugate from its site ofsynthesis to a site inside or outside of the cell membrane or cell wall.

Polypeptide conjugates can also comprise peptides added to facilitatepurification and identification of polypeptides of the invention. Suchpeptides include, for example, poly-His or the antigenic identificationpeptides described in U.S. Pat. No. 5,011,912 and in Hopp et al.,Bio/Technology 6:1204, 1988.

The invention further includes polypeptides of the invention with orwithout associated native-pattern glycosylation. Polypeptides expressedin yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells)can be similar to or significantly different from a native polypeptidein molecular weight and glycosylation pattern, depending upon the choiceof expression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Glycosyl groups can be removed through conventional methods,in particular those utilizing glycopeptidase. In general, glycosylatedpolypeptides of the invention can be incubated with a molar excess ofglycopeptidase (Boehringer Mannheim).

Correspondingly, equivalent DNA constructs that encode various additionsor substitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences are encompassed by theinvention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Known proceduresfor inactivating N-glycosylation sites in proteins include thosedescribed in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporatedby reference.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding, or substituting residues to alter Arg-Arg, Arg-Lys,and Lys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

Fragments and Uses Thereof

In yet another aspect of the invention, the polypeptides of theinvention can be subjected to fragmentation into peptides by chemicaland enzymatic means. The fragments so produced may be used for a varietyof purposes, including molecular weight markers and isoelectric pointmarkers. The polypeptides and peptide fragments can also be used for theanalysis of the degree of fragmentation. Thus, the invention alsoincludes these polypeptides and peptide fragments, as well as kits toaid in the determination of the apparent molecular weight andisoelectric point of a sample protein and kits to assess the degree offragmentation of a sample protein.

Although all methods of fragmentation are encompassed by the invention,chemical fragmentation is a preferred embodiment, and includes the useof cyanogen bromide to cleave under neutral or acidic conditions suchthat specific cleavage occurs at methionine residues (E. Gross, Methodsin Enz. 11:238-255, 1967). This can further include additional steps,such as a carboxymethylation step to convert cysteine residues to anunreactive species.

Enzymatic fragmentation is another preferred embodiment, and includesthe use of a protease such as Asparaginylendo-peptidase,Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcusaureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C underconventional conditions to result in cleavage at specific amino acidresidues. Asparaginylendo-peptidase can cleave specifically on thecarboxyl side of the asparagine residues present within the polypeptidesof the invention. Arginylendo-peptidase can cleave specifically on thecarboxyl side of the arginine residues present within thesepolypeptides. Achromobacter protease I can cleave specifically on thecarboxyl side of the lysine residues present within the polypeptides(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta660:51-55, 1981). Trypsin can cleave specifically on the carboxyl sideof the arginine and lysine residues present within polypeptides of theinvention. Enzymatic fragmentation may also occur with a protease thatcleaves at multiple amino acid residues. For example, Staphlococcusaureus V8 protease can cleave specifically on the carboxyl side of theaspartic and glutamic acid residues present within polypeptides (D. W.Cleveland, J. Biol. Chem. 3:1102-1106, 1977). Endoproteinase Asp-N cancleave specifically on the amino side of the asparagine residues presentwithin polypeptides. Endoproteinase Lys-C can cleave specifically on thecarboxyl side of the lysine residues present within polypeptides of theinvention. Other enzymatic and chemical treatments can likewise be usedto specifically fragment these polypeptides into a unique set ofspecific peptides.

Of course, the peptides and fragments of the polypeptides of theinvention can also be produced by conventional recombinant processes andsynthetic processes well known in the art. With regard to recombinantprocesses, the polypeptides and peptide fragments encompassed byinvention can have variable molecular-weights, depending upon the hostcell in which they are expressed. Glycosylation of polypeptides andpeptide fragments of the invention in various cell types can result invariations of the molecular weight of these pieces, depending upon theextent of modification. The size of these pieces can be mostheterogeneous with fragments of polypeptide derived from theextracellular portion of the polypeptide. Consistent polypeptides andpeptide fragments can be obtained by using polypeptides derived entirelyfrom the transmembrane and cytoplasmic regions, pretreating withN-glycanase to remove glycosylation, or expressing the polypeptides inbacterial hosts.

The molecular weight of these polypeptides can also be varied by fusingadditional peptide sequences to both the amino and carboxyl terminalends of polypeptides of the invention. Fusions of additional peptidesequences at the amino and carboxyl terminal ends of polypeptides of theinvention can be used to enhance expression of these polypeptides or aidin the purification of the protein. In addition, fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention will alter some, but usually not all, ofthe fragmented peptides of the polypeptides generated by enzymatic orchemical treatment. Of course, mutations can be introduced intopolypeptides of the invention using routine and known techniques ofmolecular biology. For example, a mutation can be designed so as toeliminate a site of proteolytic cleavage by a specific enzyme or a siteof cleavage by a specific chemically induced fragmentation procedure.The elimination of the site will alter the peptide fingerprint ofpolypeptides of the invention upon fragmentation with the specificenzyme or chemical procedure.

Because the unique amino acid sequence of each piece specifies amolecular weight, these pieces can thereafter serve as molecular weightmarkers using such analysis techniques to assist in the determination ofthe molecular weight of a sample protein, polypeptides or fragmentsthereof. The molecular weight markers of the invention serveparticularly well as molecular weight markers for the estimation of theapparent molecular weight of sample proteins that have similar apparentmolecular weights and, consequently, allow increased accuracy in thedetermination of apparent molecular weight of proteins.

When the invention relates to the use of fragmented peptide molecularweight markers, those markers are preferably at least 10 amino acids insize. More preferably, these fragmented peptide molecular weight markersare between 10 and 100 amino acids in size. Even more preferable arefragmented peptide molecular weight markers between 10 and 50 aminoacids in size and especially between 10 and 35 amino acids in size. Mostpreferable are fragmented peptide molecular weight markers between 10and 20 amino acids in size.

Among the methods for determining molecular weight are sedimentation,gel electrophoresis, chromatography, and mass spectrometry. Aparticularly preferred embodiment is denaturing polyacrylamide gelelectrophoresis (U. K. Laemmli, Nature 227:680-685, 1970).Conventionally, the method uses two separate lanes of a gel containingsodium dodecyl sulfate and a concentration of acrylamide between 6-20%.The ability to simultaneously resolve the marker and the sample underidentical conditions allows for increased accuracy. It is understood, ofcourse, that many different techniques can be used for the determinationof the molecular weight of a sample protein using polypeptides of theinvention, and that this embodiment in no way limits the scope of theinvention.

In addition, the polypeptides and fragmented peptides of the inventionpossess unique charge characteristics and, therefore, can serve asspecific markers to assist in the determination of the isoelectric pointof a sample protein, polypeptides, or fragmented peptide usingtechniques such as isoelectric focusing. These polypeptide or fragmentedpeptide markers serve particularly well for the estimation of apparentisoelectric points of sample proteins that have apparent isoelectricpoints close to that of the polypeptide or fragmented peptide markers ofthe invention.

The technique of isoelectric focusing can be further combined with othertechniques such as gel electrophoresis to simultaneously separate aprotein on the basis of molecular weight and charge. The ability tosimultaneously resolve these polypeptide or fragmented peptide markersand the sample protein under identical conditions allows for increasedaccuracy in the determination of the apparent isoelectric point of thesample protein. This is of particular interest in techniques, such astwo dimensional electrophoresis (T. D. Brock and M. T. Madigan, Biologyof Microorganisms 76-77 (Prentice Hall, 6^(th) ed. 1991)), where thenature of the procedure dictates that any markers should be resolvedsimultaneously with the sample protein. In addition, with such methods,these polypeptides and fragmented peptides thereof can assist in thedetermination of both the isoelectric point and molecular weight of asample protein or fragmented peptide. Polypeptides and fragmentedpeptides can be visualized using two different methods that allow adiscrimination between the sample protein and the molecular weightmarkers. In one embodiment, the polypeptide and fragmented peptidemolecular weight markers of the invention can be visualized usingantibodies generated against these markers and conventionalimmunoblotting techniques. This detection is performed underconventional conditions that do not result in the detection of thesample protein. It is understood that it may not be possible to generateantibodies against all polypeptide fragments of the invention, sincesmall peptides may not contain immunogenic epitopes. It is furtherunderstood that not all antibodies will work in this assay; however,those antibodies which are able to bind polypeptides and fragments ofthe invention can be readily determined using conventional techniques.

The sample protein is also visualized by using a conventional stainingprocedure. The molar excess of sample protein to polypeptide orfragmented peptide molecular weight markers of the invention is suchthat the conventional staining procedure predominantly detects thesample protein. The level of these polypeptide or fragmented peptidemolecular weight markers is such as to allow little or no detection ofthese markers by the conventional staining method. The preferred molarexcess of sample protein to polypeptide molecular weight markers of theinvention is between 2 and 100,000 fold. More preferably, the preferredmolar excess of sample protein to these polypeptide molecular weightmarkers is between 10 and 10,000 fold and especially between 100 and1,000 fold.

It is understood of course that many techniques can be used for thedetermination and detection of molecular weight and isoelectric point ofa sample protein, polypeptides, and fragmented peptides thereof usingthese polypeptide molecular weight markers and peptide fragments thereofand that these embodiments in no way limit the scope of the invention.

In another embodiment, the analysis of the progressive fragmentation ofthe polypeptides of the invention into specific peptides (D. W.Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977), such as byaltering the time or temperature of the fragmentation reaction, can beused as a control for the extent of cleavage of a sample protein. Forexample, cleavage of the same amount of polypeptide and sample proteinunder identical conditions can allow for a direct comparison of theextent of fragmentation. Conditions that result in the completefragmentation of the polypeptide can also result in completefragmentation of the sample protein.

As to the specific use of the polypeptides and fragmented peptides ofthe invention as molecular weight markers, the fragmentation of apreferred polypeptide of the invention with cyanogen bromide generates aunique set of fragmented peptide molecular weight markers with molecularweights of approximately 6933, 625, and 238 Daltons in the absence ofglycosylation. An additional fragment of 149 Daltons results if theinitiating methionine is present. Cleavage of SEQ ID NO:8 by cyanogenbromide generates fragments having molecular weights of 149.2, 260.3,2017.4, 3954.6, 4442.1, and 6932.7. Cleavage of SEQ ID NO:13 by cyanogenbromide generates fragments having molecular weights of 149.2, 260.3,2017.4, 3954.6, 4470.1, and 6932.7. The distribution of methionineresidues determines the number of amino acids in each peptide and theunique amino acid composition of each peptide determines its molecularweight.

In addition, the preferred isolated and purified polypeptides of theinvention (SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:13) have calculatedmolecular weights of approximately 7810 (7941 Daltons after the additionof a methionine at position 1), 17684, and 17712 Daltons, respectively,in the absence of glycosylation. The observed molecular weights of knownIL-1 polypeptides include 17, 25, 31, 33, and 35 kDa, and thus provide arange of molecular weights for use in these determinations.

Where an intact protein is used, the use of these polypeptide molecularweight markers allows increased accuracy in the determination ofapparent molecular weight of proteins that have apparent molecularweights close to 7810, 17684, or 17712 Daltons. Where fragments areused, there is increased accuracy in determining molecular weight overthe range of the molecular weights of the fragment.

Finally, as to the kits that are encompassed by the invention, theconstituents of such kits can be varied, but typically contain thepolypeptide and fragmented peptide molecular weight markers. Also, suchkits can contain the polypeptides wherein a site necessary forfragmentation has been removed. Furthermore, the kits can containreagents for the specific cleavage of the polypeptide and the sampleprotein by chemical or enzymatic cleavage. Kits can further containantibodies directed against polypeptides or fragments thereof of theinvention.

Sense and Antisense Oligonucleotides

In yet another embodiment of the invention, antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to a target mRNA sequence(forming a duplex) or to the sequence in the double-stranded DNA helix(forming a triple helix) can be made according to the invention.Antisense or sense oligonucleotides, according to the present invention,comprise a fragment of the coding region of cDNA (SEQ ID NO:5, SEQ IDNO:7, or SEQ ID NO:12). Such a fragment generally comprises at leastabout 14 nucleotides, preferably from about 14 to about 30 nucleotides.The ability to create an antisense or a sense oligonucleotide, basedupon a cDNA sequence for a given protein is described in, for example,Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of complexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortrarislation, or by other means. The antisense oligonucleotides thus canbe used to block expression of polypeptides of the invention. Antisenseor sense oligonucleotides further comprise oligonucleotides havingmodified sugar-phosphodiester backbones (or other sugar linkages, suchas those described in WO 91/06629) and wherein such sugar linkages areresistant to endogenous nucleases. Such oligonucleotides with resistantsugar linkages are stable in vivo (i.e., capable of resisting enzymaticdegradation), but retain sequence specificity to be able to bind totarget nucleotide sequences. Other examples of sense or antisenseoligonucleotides include those oligonucleotides that are covalentlylinked to organic moieties, such as those described in WO 90/10448, andother moieties that increase affinity of the oligonucleotide for atarget nucleic acid sequence, such as poly-(L-lysine). Further still,intercalating agents, such as ellipticine, and alkylating agents ormetal complexes can be attached to sense or antisense oligonucleotidesto modify binding specificities of the antisense or senseoligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides can be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).

Alternatively, sense or antisense oligonucleotides also can beintroduced into a cell containing the target nucleotide sequence byformation of a conjugate with a ligand binding molecule, as described inWO 91/04753. Suitable ligand binding molecules include, but are notlimited to, cell surface receptors, growth factors, other cytokines, orother ligands that bind to cell surface receptors. Preferably,conjugation of the ligand binding molecule does not substantiallyinterfere with the ability of the ligand binding molecule to bind to itscorresponding molecule or receptor, or block entry of the sense orantisense oligonucleotide or its conjugated version into the cell.

In yet another embodiment, a sense or an antisense oligonucleotide canbe introduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

Chromosome Mapping

In still another embodiment, oligonucleotides representing all or aportion of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:12 can be used bythose skilled in the art using well-known techniques to identify thehuman chromosome 2, and the specific locus thereof, that contains theDNA of IL-1 family members, for example, IL-1 epsilon. As set forthbelow, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12 have been mapped byradiation hybrid mapping to the long arm (2q) region of chromosome 2.That region is associated with specific diseases which include but arenot limited to glaucoma, ectodermal dysplasia, insulin-dependentdiabetes mellitus, wrinkly skin syndrome, T-cell leukemia/lymphoma,asthma, and tibial muscular dystrophy. Thus, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:12, or a fragment of these sequences can be used by oneskilled in the art using well-known techniques to study the abovedescribed diseases and other abnormalities relating to chromosome 2.This would enable one to distinguish conditions in which this marker isrearranged or deleted. In addition, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:12, or a fragment thereof can be used as a positional marker to mapother human genes of unknown location.

Therapeutic and Research Uses

Another embodiment of the invention relates to therapeutic uses of IL-1epsilon. IL-1 ligands play a central role in protection againstinfection and in promoting immune and inflammatory responses, whichincludes cellular signal transduction, activating vascular endothelialcells and lymphocytes, induction of inflammatory cytokines, acute phaseproteins, hematopoiesis, fever, bone resorption, prostaglandins,metalloproteinases, and adhesion molecules. With the continued increasein the number of known IL-1 family members, a suitable classificationscheme is one based on comparing polypeptide structure as well asfunction (activation and regulatory properties). Thus, IL-1 epsilon,like IL-1α, IL-1β, and IL-18, is likely involved in many of thefunctions noted above. In addition, IL-1 epsilon is likely involved inpromoting inflammatory responses and, therefore, may be integrallyinvolved in the causation and maintenance of inflammatory and/orautoimmune diseases such as rheumatoid arthritis, inflammatory boweldisease, and psoriasis. As such, alterations in the expression and/oractivation of IL-1 family members such as IL-1 epsilon can have profoundeffects on a plethora of cellular processes, including, but not limitedto, activation or inhibition of cell specific responses, proliferation,and inflammatory reactions based on changes in signal transduction.

Accordingly, IL-1 epsilon has therapeutic uses, such as protectingagainst infection and generating immune and inflammatory responses inindividuals whose immune and inflammatory responses are inappropriate ornonresponsive. For example, IL-1 epsilon may be useful in stimulatingthe immune system of individuals whose immune system isimmunosuppressed. Similarly, because IL-1 epsilon likely promotesinflammatory responses and is involved in the causation and maintenanceof inflammatory and/or autoimmune diseases, antagonists of IL-1 epsilonare useful in inhibiting or treating inflammatory and/or automimmunedisease.

IL-1 mediated cellular signaling often involves a molecular activationcascade, during which a receptor propagates a ligand-receptor mediatedsignal by specifically activating intracellular kinases whichphosphorylate target substrates, resulting in the activation of thetranscription factor NFKB and the protein kinases Jun N-terminal kinaseand p38 map kinase. These substrates can themselves be kinases whichbecome activated following phosphorylation. Alternatively, they can beadaptor molecules that facilitate down-stream signaling throughprotein-protein interaction following phosphorylation.

IL-1 epsilon may act as an antagonist and have an inhibitory effect onimmune responses and inflammatory responses. For example, IL-1 epsilonmay have a function similar to that of IL-1ra such that isolated andpurified IL-1 epsilon polypeptides or fragments thereof of the inventioncan be useful as therapeutic agents in inhibiting signaling and treatinginflammatory diseases and/or autoimmune diseases. However, given thedata presented in Example III, below, it is more likely that IL-1epsilon is an agonist, such as, for example IL-1α or IL-18. As statedabove, such agonists are useful in promoting immune and inflammatoryresponses in individuals whose own immune systems are inappropriatelyunder responsive. For purposes of antagonizing IL-1 epsilon activity,inhibitors of IL-1 epsilon can be engineered or designed usingtechniques known in the art. Polypeptides of the present invention,including IL-1 epsilon inhibitors, can be introduced into theextracellular environment by well-known means, such as by administeringthe protein intravenously or by coupling it to a monoclonal antibodytargeted to a specific cell type, to thereby affect signaling.

When used as a therapeutic agent, polypeptides of the invention can beformulated into pharmaceutical compositions according to known methods.The polypeptides can be combined in admixture, either as the sole activematerial or with other known active materials, with pharmaceuticallysuitable diluehts (e.g., Tris-HCl, acetate, phosphate), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,adjuvants and/or carriers. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Co. In addition, such compositions can contain thepolypeptides complexed with polyethylene glycol (PEG), metal ions, orincorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Such compositions will influence thephysical state, solubility, stability, rate of in vivo release, and rateof in vivo clearance of polypeptides of the invention.

The dosage of the composition can be readily determined by those ofordinary skill in the art. The amount to be administered and thefrequency of administration can be determined empirically and will takeinto consideration the age and size of the patient being treated, aswell as the malady being treated.

Treatment comprises administering the composition by any method familiarto those of ordinary skill in the art, including intravenous,intraperitoneal, intracorporeal injection, intra-articular,intraventricular, intrathecal, intramuscular, subcutaneous, topically,tonsillar, intranasally, intravaginally, and orally. The composition mayalso be given locally, such as by injection into the particular area,either intramuscularly or subcutaneously.

In addition, the DNA, polypeptides, and antibodies against polypeptidesof the invention can be used as reagents in a variety of researchprotocols. A sample of such research protocols are given in Sambrook etal. Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Vol. 1-3, ColdSpring Harbor Laboratory Press, (1989). For example, these reagents canserve as markers for cell-specific or tissue-specific expression of RNAor proteins. Similarly, these reagents can be used to investigateconstitutive and transient expression of RNA or polypeptides. As notedabove, the DNA can be used to determine the chromosomal location of DNAand to map genes in relation to this chromosomal location. The DNA canalso be used to examine genetic heterogeneity and heredity through theuse of techniques such as genetic fingerprinting, as well as to identifyrisks associated with genetic disorders. The DNA can be further used toidentify additional genes related to the DNA and to establishevolutionary trees based on the comparison of sequences. The DNA andpolypeptides can be used to select for those genes or proteins that arehomologous to the DNA or polypeptides, through positive screeningprocedures such as Southern blotting and immunoblotting and throughnegative screening procedures such as subtraction.

Further, because IL-1 epsilon is a ligand, it takes part inprotein-protein interactions with at least one or more proteins, i.e.its receptor(s). Thus, the polypeptides and fragments of the inventioncan be used as reagents to identify (a) proteins that the polypeptideregulates, and (b) proteins with which it might interact.

Therefore, IL-1 epsilon ligands or polypeptides comprising portions ofan IL-1 epsilon ligand could be used by coupling recombinant protein toan affinity matrix, or by using them as “baits” in the yeast 2-hybridsystem according to well established molecular biology techniques, toidentify proteins that interact directly with the polypeptide of theinvention. Further, the IL-1 epsilon polypeptides and fragments of thepresent invention find use in studies directed toward discovering IL-1receptors and/or IL-1 epsilon receptors. For example, IL-1 epsilonpolypeptides and IL-1 epsilon polypeptide fragments can be used inbinding studies to identify receptor-expressing cells. Suitable bindingstudies are known in the art and are well within the knowledge of thoseskilled in the art. Similarly, the IL-1 epsilon polypeptides andpolypeptide fragments of the present invention find additional uses incloning receptors using expression cloning techniques.

The polypeptides and fragments thereof can also be used as reagents inthe study of signaling pathways utilized by IL-1 and IL-1R homologs orfamily members, and/or in blocking those signaling pathways. Such novelIL-1 receptor homologs can be specifically used as reagents to identifynovel molecules involved in signal transduction pathways, characterizecell and tissue expression, understand their roles in development,immune, and inflammatory responses, and identify regulatory moleculesand physiologically relevant protein substrates.

Alternatively, polypeptides of the invention could be engineered priorto expression with a tag such as poly-His or FLAG, then be expressed andpurified using either nickel chelate chromatography or anti-FLAGantibody coupled to a resin, respectively. Once bound to the resin, thepolypeptide of the invention could be covalently attached using abifunctional cross-linking agent using well established techniques. Thecovalently bound polypeptide to the resin could then be used to purifymolecules from cell lysates or cell supernatants (following treatmentwith various reagent) through their affinity for the polypeptide of theinvention.

Antibodies

Within the therapeutic and research aspects of the invention,polypeptides of the invention, and peptides based on the amino acidsequence thereof, can be utilized to prepare antibodies thatspecifically bind to the polypeptides. The term “antibodies” is meant toinclude polyclonal antibodies, monoclonal antibodies, fragments thereofsuch as F(ab′)₂, and Fab fragments, as well as any recombinantlyproduced binding partners. Antibodies are defined to be specificallybinding if they bind polypeptides of the invention with a K_(a) ofgreater than or equal to about 10⁷ M⁻¹. Affinities of binding partnersor antibodies can be readily determined using conventional techniques,for example those described by Scatchard et al., Ann. N. Y Acad. Sci.,51:660 (1949).

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice, or rats, using procedures that are well-known in the art.In general, purified polypeptides of the invention, or a peptide basedon the amino acid sequence of polypeptides of the invention that isappropriately conjugated, is administered to the host animal typicallythrough parenteral injection. The immunogenicity of these polypeptidescan be enhanced through the use of an adjuvant, for example, Freund'scomplete or incomplete adjuvant. Following booster immunizations, smallsamples of serum are collected and tested for reactivity to thepolypeptides. Examples of various assays useful for such determinationinclude those described in: Antibodies: A Laboratory Manual, Harlow andLane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well asprocedures such as countercurrent immuno-electrophoresis (CIEP),radioimmunoassay, radio-immunoprecipitation, enzyme-linkedimmuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, seeU.S. Pat. Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies can be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKeam, and Bechtol (eds.), 1980. Briefly, the host animals,such as Balb/c mice are injected intraperitoneally at least once, andpreferably at least twice at about 3 week intervals with isolated andpurified polypeptides or conjugated polypeptides of the invention,optionally in the presence of adjuvant. 10 μg of isolated and purifiedpolypeptide of the invention or peptides based on the amino acidsequence of polypeptides of the invention in the presence of RIBIadjuvant (RIBI Corp., Hamilton, Mont.). Mouse sera are then assayed byconventional dot blot technique or antibody capture (ABC) to determinewhich animal produces the highest level of antibody and whose spleencells are the best candidate for fusion. Approximately two to threeweeks later, the mice are given an intravenous boost of the polypeptidesor conjugated polypeptides such as 3 μg suspended in sterile PBS. Miceare later sacrificed and spleen cells fused with commercially availablemyeloma cells, such as Ag8.653 (ATCC), following established protocols.Briefly, the myeloma cells are washed several times in media and fusedto mouse spleen cells at a ratio of about three spleen cells to onemyeloma cell. The fusing agent can be any suitable agent used in theart, for example, polyethylene glycol (PEG) or more preferably, 50% PEG:10% DMSO (Sigma). Fusion is plated out into, for example, twenty 96-wellflat bottom plates (Corning) containing an appropriate medium, such asHAT supplemented DMEM media and allowed to grow for eight days.Supernatants from resultant hybridomas are collected and added to, forexample, a 96-well plate for 60 minutes that is first coated with goatanti-mouse Ig. Following washes, ¹²⁵I-polypeptide or peptides of theinvention are added to each well, incubated for 60 minutes at roomtemperature, and washed four times. Positive wells can be subsequentlydetected by conventional methods, such as autoradiography at −70EC usingKodak X-Omat S film. Positive clones can be grown in bulk culture andsupernatants are subsequently purified, such as over a Protein A column(Pharmacia). It is understood of course that many techniques could beused to generate antibodies against polypeptides and fragmented peptidesof the invention and that this embodiment in no way limits the scope ofthe invention.

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7:394 (1989).

Other types of “antibodies” can be produced using the informationprovided herein in conjunction with the state of knowledge in the art.For example, antibodies that have been engineered to contain elements ofhuman antibodies that are capable of specifically binding polypeptidesof the invention are also encompassed by the invention.

Once isolated and purified, the antibodies against polypeptides of theinvention can be used to detect the presence of the polypeptides in asample using established assay protocols. Further, the antibodies of theinvention can be used therapeutically or for research purposes to bindto the polypeptides and inhibit its activity in vivo or in vitro.

Antibodies immunoreactive with polypeptides of the invention, and inparticular, monoclonal antibodies against these polypeptides, are nowmade available through the invention. Such antibodies can be useful forinhibiting polypeptide activity in vivo and for detecting the presenceof polypeptides of the invention in a sample.

In another embodiment, antibodies generated against a polypeptide andfragmented peptides of the invention can be used in combination withpolypeptide or fragmented peptide molecular weight markers of theinvention to enhance the accuracy in the use of these molecular weightmarkers to determine the apparent molecular weight and isoelectric pointof a sample protein. Polypeptide or fragmented peptide molecular weightmarkers of the invention can be mixed with a molar excess of a sampleprotein and the mixture can be resolved by two dimensionalelectrophoresis by conventional means. Polypeptides can be transferredto a suitable protein binding membrane, such as nitrocellulose, byconventional means and detected by the antibodies of the invention.

Drug Discovery

The purified polypeptides according to the invention will facilitate thediscovery of inhibitors of such polypeptides. The use of a purifiedpolypeptide of the invention in the screening of potential inhibitorsthereof is important and can eliminate or reduce the possibility ofinterfering reactions with contaminants.

In addition, polypeptides of the invention can be used forstructure-based design of polypeptide-inhibitors. Such structure-baseddesign is also known as “rational drug design.” The polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of the polypeptide structural information inmolecular modeling software systems to assist in inhibitor design andinhibitor-polypeptide interaction is also encompassed by the invention.Such computer-assisted modeling and drug design can utilize informationsuch as chemical conformational analysis, electrostatic potential of themolecules, protein folding, etc. For example, most of the design ofclass-specific inhibitors of metalloproteases has focused on attempts tochelate or bind the catalytic zinc atom. Synthetic inhibitors areusually designed to contain a negatively-charged moiety to which isattached a series of other groups designed to fit the specificitypockets of the particular protease. A particular method of the inventioncomprises analyzing the three dimensional structure of polypeptides ofthe invention for likely binding sites of substrates, synthesizing a newmolecule that incorporates a predictive reactive site, and assaying thenew molecule as described above.

The following examples are presented to promote a fuller understandingof this invention. These examples do not, however, limit the scope ofthe invention.

EXAMPLE I Isolation and Identification of a New Human IL-1 Ligand

We screened a human genomic phage library (Stratagene catalog # 946205)using a mixture of ³²P-labeled single-strand DNA probes corresponding tothe entire coding sequence of murine IL-1 epsilon. After low stringencywashing (low stringency washing is defined as 0.2×SSC/0.1% SDS, at roomtemperature, Ausubel et al. Current Protocols in Molecular Biology, Vol.2, p. 10.3, John Wiley & Sons, Inc., (1996)), a positive clone with astrong hybridization signal was identified. DNA made from this clone andsubjected to Southern analysis identified a 5.5 kb Sal I-Asp 718restriction fragment which was subcloned into pBluescript and sequenced.Homology analysis of the 5.5 kb fragment using the UWGCG computerprogram “bestfit” revealed that a 212 bp region within the clone was 74%similar at the nucleotide level to the 3 prime exon of murine IL-1epsilon. As set forth in FIG. 3, this 212 bp sequence contains an openreading frame of 70 amino acids with 66% similarity (64% identity) tothe 3 prime exon of murine IL-1 epsilon.

The genomic sequence around the human IL-1 epsilon locus was extendedanother 5 kb in the 5′ direction using a Genome Walking kit (availablefrom Clonetech) in accordance with manufacturer's instructions. Analysisof the sequence of this upstream region revealed three additionalputative exons. RT-PCR was used to confirm the expression of theseexons, and their linkage into a single IL-1 epsilon cDNA, in RNA fromfour different human tissue sources (thymus, tonsil, and the cell linesHL-60 and THP-1). Additionally, a cDNA clone was obtained from theStratagene Universal Human cDNA Library Array I that also demonstratedthe joining of the three 3′-most exons. The cDNA clone from theUniversal Human cDNA Library Arry I was a partial clone that did notextend to the 5′ end of the open reading frame. Full-length human IL-1epsilon DNA sequences are disclosed in SEQ ID NO:7 and SEQ ID NO:12. Thepolypeptides encoded by SEQ ID NO:7 and SEQ ID NO:12 are disclosed inSEQ ID NO:8 and SEQ ID NO:13, respectively. As set forth in FIG. 4,amino acids 51-159 of SEQ ID NO:8 and SEQ ID NO:13 share 53% similarity(49% identity) with the murine IL-1 epsilon (long form).

EXAMPLE II Chromosome Mapping of Human IL-1 Epsilon by Radiation HybridMapping

PCR was performed using the Whitehead Institute/MIT Center for GenomeResearch Genebridge4 panel of 93 radiation hybrids(http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/rhmap/genebridge4.html).Primers were used which lie within the putative 3 prime exon of humanIL-1 epsilon and which amplify a 195 bp product from human genomic DNA,but do not amplify hamster genomic DNA. The results of the PCRs wereconverted into a data vector that was submitted to the Whitehead/MITRadiation Mapping site on the internet (http://www-seq.wi.mit.edu). Thedata was scored and the chromosomal assignment and placement relative toknown Sequence Tag Site (STS) markers on the radiation hybrid map wasprovided. According to the results, human IL-1 epsilon lies onchromosome 2, at 11.54 cR from STS D2S121 and 4.3 cR from the markerCHLC.GAAT11C03. The following web site provides additional informationabout radiation hybrid mapping:http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.1NTRO.html).

EXAMPLE III Activation of Signaling Molecules in Human Cells by HumanIL-1 Epsilon

The following describes tests and results that were carried out todetermine whether Il-1 epsilon is capable of activating some of the samesignaling molecules involved in stress responses as are activated byIL-1α, IL-1β and other inflammatory cytokines.

Human IL-1 epsilon was transfected into COS-1 cells. Several days afterthe transfection, conditioned medium (containing the transientlyexpressed IL-1 epsilon) was harvested. Test cells were incubated withthis conditioned medium, or alternatively with conditioned medium fromCOS-1 cells transfected with the empty expression vector. Approximately10 minutes following the incubation, cell extracts were prepared fromthe test cells, and the presence of activated signaling molecules wasassayed by the use of antibodies specific for the phosphorylated formsof IKBα (phosphorylation on Ser32), p38 MAP kinase (phosphorylation onThr180 and Tyr182), and Stress-Activated Protein Kinase (SAPK/JNK)(phosphorylation on Thr183/Tyr185) (the antibodies were obtained fromNew England Biolabs, Beverly, Mass.). These signal transductionmolecules are known to be involved in a wide range of cellular responsesto stimuli such as UV irradiation, endotoxin, and inflammatory cytokinesincluding IL-1β. Compared to control conditioned medium, conditionedmedium containing human Il-1 epsilon activated IKBα and p38 MAP kinasephosphorylation in a number of human cell lines including Human ForeskinFibroblasts and Human Umbelical Vein Endothelial Cells (ATCC CRL-1730).In the non-Hodgkins lymphoma cell line K299, human IL-1 epsilonspecifically activated JNK/SAPK phosphorylation. These resultsdemonstrate that IL-1 epsilon is involved in stress response signalingpathways.

EXAMPLE IV Tissue Distribution of Human IL-1 Epsilon

The tissue distribution of human IL-1 epsilon mRNA was investigatedusing PCR amplification from a panel of first strand cDNA templates.Specifically, a Clontech (Palo Alto, Calif.) Human Multiple Tissue cDNAPanel was screened using a forward primer in exon 2 and a reverse primerin exon 4, which, together, amplify a 450 base-pair fragment of IL-1epsilon. The PCR reaction was run for 35 cycles with an annealingtemperature of 60EC. PCR products were detected on an agarose gel usingethidium bromide.

Human IL-1 epsilon mRNA was detected in the spleen, lymph node, thymus,tonsil, and leukocyte tissues. The tissue with the highest levels ofhuman IL-1 epsilon mRNA is tonsil.

EXAMPLE V Activation of ICAM-1 Levels in Human Cells by Human IL-1Epsilon

The following describes tests and results that were carried out todetermine whether Il-1 epsilon is capable of activating some of the samecell surface molecules involved in stress responses as are activated byIL-1α, IL-1β and other inflammatory cytokines.

Human IL-1 epsilon was transfected into COS-1 cells. Several days afterthe transfection, conditioned medium (containing the transientlyexpressed IL-1 epsilon) was harvested. Human foreskin fibroblast (HFF)cells were incubated for 18 hours at 37EC with this conditioned mediumdiluted 1:1 with fresh 0.5% serum-containing medium, or alternativelywith conditioned medium from control COS-1 cells transfected with theempty expression vector, diluted 1:1 with fresh 0.5% serum-containingmedium.

Following treatment with the conditioned medium from COS-1 cells, theHFF cells were washed twice with PBS and removed from the tissue culturevessel with versene (non-trypsin reagent). Cell-surface ICAM-1 levelswere measured by staining with anti-CD54-PE antibody (Pharmingen, SanDiego, Calif.) on ice for one hour followed by washing and FACS-baseddetection.

HFF cells incubated in conditioned medium from control COS-1 cellsexhibited a slight increase in ICAM-1 levels, relative to untreatedcells. On the other hand, HFF cells that were treated with conditionedmedium from COS-1 cells that had been transfected with IL-1 epsilonexhibited a two-fold increase in cell-surface ICAM-1 levels. The levelof ICAM-1 staining seen on the IL-1 epsilon treated HFF cells wascomparable to that induced on the same cells by purified IL-1∃.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, which arehereby incorporated by reference.

The embodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan recognizes many otherembodiments are encompassed by the claimed invention.

1. An antibody that binds to a polypeptide selected from the groupconsisting of: (a) a polypeptide of SEQ ID NO:8: (b) a polypeptide ofSEQ ID NO:13; (c) a polypeptide that is at least 90% identical to SEQ IDNO:8, wherein the polypeptide activates IKBα or p38 MAP kinasephosphorylation or cell surface expression of ICAM-1; (d) a polypeptidethat is at least 90% identical to SEQ ID NO:13, wherein the polypeptideactivates IKBα or p38 MAP kinase phosphorylation or cell surfaceexpression of ICAM-1; (e) a polypeptide encoded by a DNA comprising apolynucleotide of SEQ ID NO:5, (f) a polypeptide encoded by a DNAcomprising a polynucleotide of SEQ ID NO:7, and (g) a polypeptideencoded by a DNA comprising a polynucleotide of SEQ ID NO:12.
 2. Theantibody of claim 1 that binds to the polypeptide of SEQ ID NO:8.
 3. Theantibody of claim 1 that binds to the polypeptide of SEQ ID NO:13. 4.The antibody of claim 1, wherein the antibody is a monoclonal antibody.5. The antibody of claim 2, wherein the antibody is a monoclonalantibody.
 6. The antibody of claim 3, wherein the antibody is amonoclonal antibody.